Cat-mouse heterohybridoma and gene fragment coding for constant region of feline immunoglobulin

ABSTRACT

A Gene fragment which comprises a DNA sequence coding for an amino acid sequence of a constant region of feline immunoglobulin λ chain; a gene fragment which comprises a DNA sequence coding for an amino acid sequence of a constant region of feline immunoglobulin κ chain; a gene fragment which comprises a DNA sequence coding for the constant region of feline immunoglobulin γ chain; a recombinant DNA molecule coding for an amino acid sequence of a mouse-cat chimeric antibody which comprises a gene fragment coding for an amino acid sequence of a variable region of a mouse immunoglobulin and a gene fragment coding for an amino acid sequence of a constant region of a feline immunoglobulin wherein the latter gene fragment is linked to the 3&#39; site of the former gene fragment; a polypeptide of a mouse-cat chimeric antibody which is expressed from a cell transformed with an expression vector for cells wherein said recombinant DNA molecule coding for an amino acid sequence of the mouse-cat chimeric antibody is incorporated; a cat-mouse heterohybridoma which produces feline immunoglobulin; and a process for preparing a feline immunoglobulin gene.

This application is a continuation of U.S. application Ser. No.07/565,233 filed Aug. 10, 1990 now abandoned.

The present invention relates to a novel feline monoclonal antibodywhich is expected to be useful for diagnosis, treatment and preventionof feline diseases, especially feline infectious diseases. Moreparticularly, it relates to a novel cat-mouse heterohybridoma capable ofproducing a feline immunoglobulin, a gene fragment coding for a constantregion of feline immunoglobulin and a feline chimeric antibody producedby utilizing said gene fragment.

TECHNICAL BACKGROUND AND PRIOR ART

From ancient times, cats have been favorably treated as a pet by humans.In modern Europe and America, they are called "Companion species" andnow becoming a member of human society. On the other hand, cats havehitherto been used as an experimental animal in various fields includingmedicine, pharmacy, animal husbandry, veterinary, psychology etc. Theyare also used as a minimal disease cat in recent years in tests fordetermination of effect and safety of drugs, and hence, usefulnessthereof for humans becoming greater and greater. In any case, it isearnestly desired to establish a method for more certain diagnosis,treatment and prevention of feline diseases, especially felineinfectious diseases in order to keep cats in healthy conditions.

There are many feline viral diseases, and among them, those caused byfeline rhinotracheitis virus, feline parvovirus, feline infectiousperitonitis, etc. are acute diseases having a very high lethality rate.Although vaccines for prevention of these diseases have been developed,only symptomatic therapy such as by antibiotics and sulfonamides whichprevents secondary bacterial infections has been available for treatingthose cats infected and attacked with these diseases, and hence, theconventional methods for treating these diseases are still insufficient.

Hitherto, a hyperimmune serum and an immunoglobulin derived from serumhave been utilized for treatment of these diseases and confirmed to beeffective. However, with the popularity of the idea for kindly treatmentof animals, feline serum materials have become hard to obtain, andhence, this treatment can not be used nowadays. Therefore, developmentof a monoclonal antibody capable of neutralizing the infected viruses inplace of the conventional hyperimmune serum will greatly contribute tothe treament of these viral diseases.

As mentioned above, a monoclonal antibody having a neutralizing activityagainst viruses can be used as the alternative to the hyperimmune serum.Hitherto, basic techniques of preparing monoclonal antibodies have beenestablished mainly for a mouse monoclonal antibody. Monoclonalantibodies produced by cells such as hybridomas can advantageously beobtained in a large amount and semipermanently and solve the problem ofmaterial insufficiency. However, the monoclonal antibody in this caseshould be a feline monoclonal antibody instead of the conventional mousemonoclonal antibody in order to eliminate side effects such asanaphylatic shock, serum disease, etc. caused by the use in cats of themouse monoclonal antibody which acts as a heteroprotein to cats.

Methods for preparing such feline monoclonal antibody as a drug fortreating the feline viral diseases include:

(1) a method using a cat--cat hybridoma;

(2) a method using a feline lymphocyte transformed with some viral orchemical agent;

(3) a method using a cat-mouse heterohybridoma;

(4) a method using a cat-(cat-mouse) hybridoma derived from a cat-mouseheterohybridoma; and

(5) a method by gene recombination techniques of a mouse (V)-cat (C)chimeric monoclonal antibody wherein a variable (V) region which bindsto an antigen is derived from a mouse monoclonal antibody havingneutralizing activity against viruses and a constant (C) region which isresponsible for antigenicity, immunogenicity and physiological activityis derived from a feline monoclonal antibody.

However, none of the above methods have hitherto been reported to beeffectively used.

In the method (1), a fusion efficiency is quite low and no appropriatemyeloma strain is available. In case of the method (2), there are noappropriate virus corresponding to EB virus in case of human and noappropriate chemical agents. The methods (3) and (4) will have muchdifficulty (for example, a stability problem etc.) in obtaining thedesired feline monoclonal antibody with high efficiency in view of thecase of preparation of a human monoclonal antibody. Therefore, it isexpected that the method (5) using the chimeric monoclonal antibody isthe most realizable method among these five methods.

The chimeric monoclonal antibody is prepared by incorporating a plasmidvector containing a mouse (V)-cat (C) chimeric antibody gene into ananimal host cell (e.g. mouse myeloma cell), expressing said gene in thehost cell and collecting the monoclonal antibody from a supernatant ofthe culture, wherein said mouse (V)-cat (C) chimeric antibody gene issuch that a V (variable) gene is cloned from a mouse--mouse hybridomacapable of producing a mouse monoclonal antibody as a source of a genecoding for a V region, a C (constant) gene is cloned from a feline cellsuch as a feline antibody-producing cell capable of producing a felinemonoclonal antibody as a source of a gene coding for a C region and saidV gene and said C gene are linked to each other. Several reports arefound as to human chimeric antibodies (Japanese Patent FirstPublications Nos. 155132/1985 and 47500/1986).

As mentioned above, a gene coding for an amino acid sequence in avariable (V) region of an antibody molecule capable of binding to adesired antigen and a gene coding for an amino acid sequence in aconstant (C) region of a feline immunoglobulin are required forpreparing the feline chimeric antibody. The gene coding for the variable(V) region of the chimeric antibody is derived from a cell capable ofproducing a mouse monoclonal antibody having a neutralizing activityagainst the above mentioned various feline viruses and said cell can beprepared rather easily by the conventional mouse--mouse hybridomaproducing procedure. However, the gene coding for the constant (C)region of the chimeric antibody, i.e. the gene coding for the constant(C) region of the feline immunoglobulin is still unknown in itsstructure and has never been cloned. Therefore, in order to prepare thefeline chimeric antibody, it is inevitably required to find the genecoding for the amino acid sequence of the constant (C) region of thefeline immunoglobulin.

In addition, although there is much difficulty in obtaining themonoclonal antibody showing a desired specificity in case of the methods(1) to (4), materials (cell strains) effective for preparing thechimeric antibody can effectively be provided in case of the method (5)since any cell which produces the feline immunoglobulin regardless ofits specificity can preferably be employed as material for providing agene coding for the C region of the feline immunoglobulin for preparingthe chimeric antibody.

BRIEF SUMMARY OF THE INVENTION

Under the circumstances, the present inventors have succeeded inpreparing cat-mouse hybridomas which produce the feline immunoglobulin,in isolating a gene coding for the amino acid sequence in the constant(C) region of the feline immunoglobulin using said hybridoma cells andfeline cells, and in preparing a feline chimeric antibody using the genefragment coding for the constant (C) region of the felineimmunoglobulin.

An object of the invention is to provide a hitherto unreported cat-mouseheterohybridoma capable of producing the feline immunoglobulin and ahitherto genetically unanalyzed DNA fragment coding for an amino acidsequence in the constant (C) region of the feline immunoglobulin.Another object of the invention is to provide a process for preparingthe feline chimeric antibody using said DNA fragment coding for theamino acid sequence in the constant (C) region of the felineimmunoglobulin, said chimeric antibody being useful in agents fordiagnosis, treatment and prevention of feline diseases, especiallyinfectious feline diseases, without showing side effects. These andother objects and advantages of the invention will be apparent to thoseskilled in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of EIA using an anti-feline antibody provingthat IgG produced by the cat-mouse hybridomas prepared by the presentinvention is a feline monoclonal antibody;

FIG. 2 shows the results of Western blot analysis using an anti-felineantibody proving that IgG produced by the cat-mouse hybridomas preparedby the present invention is a feline monoclonal antibody;

FIG. 3 shows a restriction enzyme map of a DNA fragment (T1-62) codingfor the constant region of feline immunoglobulin λ chain cloned by thepresent invention and the regions (→) in which a nucleotide sequence wasanalyzed;

FIG. 4 shows the results of Southern hybridization analysis where a DNAfragment (T1-62) coding for the constant region of feline immunoglobulinλ chain cloned by the present invention is hybridized with a chromosomalDNA (EcoRI digestion) of feline hepatocyte;

FIG. 5 shows the results of Northern hybridization analysis where a DNAfragment (T1-62) coding for the constant region of feline immunoglobulinλ chain cloned by the present invention is hybridized with poly A+RNA ofFM-T1 cells (lane 1) or poly A+RNA of FM-T2 cells (lane 2);

FIG. 6 shows a nucleotide sequence coding for the constant region offeline immunoglobulin λ chain present in the DNA fragment (T1-62) clonedby the present invention;

FIG. 7 shows a whole amino acid sequence of the constant region offeline immunoglobulin λ chain coded in the DNA fragment (T1-62) clonedby the present invention;

FIG. 8 shows the results of Southern hybridization analysis where anEcoRI fragment of a chromosomal DNA of feline hepatocyte is hybridizedwith ³² P!-labelled probe containing human Cκ chain region;

FIG. 9 shows the results of Northern hybridization analysis where polyA+RNA extracted from feline spleen cells is hybridized with a ³²P!-labelled CEκ8a probe;

FIG. 10 shows a restriction enzyme map of a DNA fragment (CEκ8a) codingfor the constant region of feline immunoglobulin κ chain cloned by thepresent invention and the regions (→) in which a nucleotide sequence wasanalyzed;

FIG. 11 shows a nucleotide sequence coding for the constant region offeline immunoglobulin κ chain present in the DNA fragment CEκ8a clonedby the present invention;

FIG. 12 shows a whole amino acid sequence of the constant region offeline immunoglobulin κ chain coded in the DNA fragment CEκ8a cloned bythe present invention;

FIG. 13 shows the results of Southern hybridization analysis where aBamHI fragment of a chromosomal DNA of feline hepatocyte is hybridizedwith a ³² P!-labelled probe containing the human Cγl chain region;

FIG. 14 shows a restriction enzyme map of the chromosomal DNA fragment(CB25γ7c) coding for the constant region of feline immunoglobulin γchain cloned by the present invention;

FIG. 15 shows the result of Northern hybridization analysis where polyA+RNA extracted from feline antibody-producing heterohybridoma FM-T1 ishybridized with a ³² P!-labelled CB25γ7c probe;

FIG. 16 shows a restriction enzyme map of a cDNA fragment T1CB1a clonedby the present invention and the regions (→) in which a nucleotidesequence was analyzed;

FIG. 17 shows a nucleotide sequence coding for the constant region offeline immunoglobulin γ chain present in a cDNA fragment T1CB1a clonedby the present invention;

FIG. 18 shows a whole amino acid sequence of the constant region offeline immunoglobulin γ chain coded in a cDNA fragment T1CB1a cloned bythe present invention;

FIG. 19 shows a restriction enzyme map of a clone JP2gL411 containing Vκregion gene isolated from anti-CPV mouse monoclonal antibody-producingcells;

FIG. 20 shows nucleotide and amino acid sequences of a Vκ region geneJP2gL411 isolated from anti-CPV mouse monoclonal antibody-producincells; and

FIG. 21 shows a construction of a gene (pSV2-EPLCCκ) expressing an Lchain of an anti-CPV mouse-cat chimeric antibody.

DETAILED DESCRIPTION OF THE INVENTION

There are two approaches for isolating the gene coding for the aminoacid sequence in the constant (C) region of the feline immunoglobulin.That is, the first is to construct a library from a chromosomal DNA infeline cells and then to clone the gene coding for the amino acidsequence in C region according to the conventional procedure forexample, see T. Maniatis "Molecular Cloning" Cold Spring Harbor Lab.(1982)!, and the second is to construct a library by synthesizing cDNAfrom a messenger RNA (mRNA) of feline cells and then to clone the Cregion gene according to the conventional procedure for example, see"DNA cloning Vol. I" ed. by D. M. Glover, IRL press (1985)!.

For the screening procedure, there can be used mainly four processes;i.e. (1) a process which comprises purifying the antibody proteinproduced by the feline antibody-producing cell, analyzing the amino acidsequence of said protein, and synthesizing a nucleotide sequencecorresponding to said amino acid sequence, and then using saidnucleotide sequence as a probe for screening (hybridization); (2) aprocess by cross-hybridization using a probe synthesized by referring toa gene fragment of mouse and human immunoglobulin genes or nucleotidesequences thereof previously reported for example, Sakano et al.,Nature, 286, p676 (1980); E. E. Max et al., J. Biol. Chem., 256, p5116(1981); J. W. Ellison et al., Nuc. Acids. Res., 10, p4071 (1982); and P.A. Heiter et al., Cell, 22, p197 (1980)!; (3) a process which comprisessynthesizing a primer based on a nucleotide sequence on the analogy ofan amino acid sequence of a feline antibody protein or a nucleotidesequence on the analogy of the mouse and human immuno-globulin genespreviously reported and then screening based on the primer using the PCRprocedure R. Orlandi et al., Proc. Natl. Acad. Sci. USA, 86, p3833(1989)!; and (4) a process which comprises expressing the felineantibody gene incorporated into an expression vector (e.g. λgt11) in E.coli or in an animal cell and screening the expression product by usingan antiserum (or a monoclonal antibody) directed to the feline antibodyprotein. In case of the cDNA cloning procedure, all the above processes(1) to (4) can be used and in case of the chromosomal DNA cloningprocedure the processes (1) to (3) can be used.

In any case, the screening process (1) using the feline antibodyprotein-producing cells is preferable in view of the cloning efficiencyand is essential in case of the above second process for isolating thegene using the cDNA synthesized from messenger RNA. Although it ispossible to use polyclonal cells such as spleen cells or lymph nodecells as the antibody-producing cells, monoclonal cells are preferablein view of easiness of genetic analysis.

There are several methods for establishing the monoclonalantibody-producing cells as cited above (1) to (5). However, the methods(1) and (2) are extremely difficult to practice and the method (4)requires the cat-mouse heterohybridomas of the method (3). Inconclusion, it is most important to obtain the cat-mouseheterohybridomas of the method (3).

The cat-mouse heterohybridomas can be prepared by the several methodspreviously disclosed. According to these methods, the present inventorshave established cat-mouse heterohybridomas FM-T-1, -T2 and -T3 whichproduce the feline monoclonal antibody. Among these cat-mouseheterohybridomas, FM-T1 is the most preferable cell for preparing thegene of the present invention and has been deposited at the FermentationResearch Institute Agency of Industrial Science and Technology, Japanunder Budapest Treaty under the accession number FERM BP-2947.

As mentioned above, the present inventors have cloned several kinds ofgene fragments coding for the constant region of immunoglobulin fromfeline cells. The cloned gene fragments coding for the C region offeline immunoglobulin of the present invention were analyzed bycomparing an amino acid sequence deduced from the nucleotide sequencethereof with the corresponding sequences of the C region genes ofimmunoglobulin from other animal species, and as a result, it has beenfound that each gene fragment of the present invention contains genefragments coding for λ chain, κ chain and γ chain, respectively.

λ Chain of immunoglobulin has already been found in human P. A. Hieteret al., Nature, 294, p536 (1981); G. F. Hollis et al., Nature 296, p321(1982)! and in mouse B. Blomberg et al., Proc. Natl. Acad. Sci. USA, 79,p530 (1982); J. Miller et al., Nature, 295, p428 (1982)! and has alsobeen reported for other animal species such as rabbit Duvoisin, MR. M.et al., J. Immunol., 136, p4297-4302 (1986)!. However, there has neverbeen reported the feline λ chain of the present invention, the aminoacid sequence thereof and the nucleotide sequence coding therefore.

The cDNA fragment coding for the constant region of felineimmunoglobulin λ chain prepared as mentioned above has been analyzed forits nucleotide sequence. Then, the amino acid sequence of said constantregion has been deduced from the nucleotide sequence and compared withthe amino acid sequences of the constant region of immunoglobulin λchain derived from human, mouse, rabbit, etc. previously reported. As aresult, it has been found that the constant region of felineimmunoglobulin λ chain has a specific amino acid sequence at theN-terminal region of the polypeptide of said λ chain constant regionwhich has the following amino acid sequence (A) at the N-terminal regionof the first cysteine counted from the N-terminus of said polypeptide:

    -Ser-Ala-xxx-xxx-xxx-xxx-xxx-xxx-Cys-                      (A)

wherein xxx means an optional amino acid residue.

From comparison among the amino acid sequence of the constant region offeline immunoglobulin λ chain analyzed in accordance with the presentinvention, the amino acid sequence of the constant region of canineimmuno-globulin λ chain separately analyzed by the present inventors andvarious amino acid sequences of the constant region of immunoglobulin λchain derived from several animal species, it has been found that theregion -Ser-Ala- present at the N-terminus of the above cysteine is aregion whose amino acid sequence varies with animal species such cat,mouse, human, etc. In addition, it has been found that this region isquite well preserved among subclasses, for example, as an amino acidsequence of the constant region of human λ chain, and the above sequence(A) is supposed to be specific for the constant region of the felineimmunoglobulin λ chain. The region cloned by the present invention hasthe following amino acid sequence (A') which is one example of thepreferable specific amino acid sequences present in the constant regionof the feline immunoglobulin λ chain.

    -Ser-Ala-Asn-Lys-Ala-Thr-Leu-Val-Cys-                      (A')

The cat-mouse heterohybridoma of the present invention produces felineimmunoglobulin containing a peptide of a λ chain C region having theabove amino acid sequence (A) or (A'). The gene fragment coding for theconstant region of the feline immunoglobulin λ chain of the presentinvention also contains a DNA fragment coding for the above amino acidsequnece (A) or (A'). Such amino acid sequence contained in the above λchain is believed to be an important amino acid sequence for determiningthe C region of the feline immunoglobulin λ chain and is firstlydisclosed by the present invention. In the constant region polypeptideof the feline λ chain, the 11th-9th amino acid sequence at theN-terminal region of from the second cysteine residue counted from theC-terminus is deemed to be the region whose amino acid sequence varieswith species such as cat, dog, etc. as mentioned above, and it is foundthat the constant region of the feline immunoglobulin λ chain of thepresent invention has the corresponding specific sequence of-Pro-Asn-Glu-. One preferable example of the gene coding for theconstant region of the feline immunoglobulin λ chain is a gene fragmentcoding for the amino acid sequnece as shown in FIG. 7 and one example ofthe nucleotide sequence of said gene is as shown in FIG. 6.

On the analogy of the case of human and mouse P. A. Hieter et al.,Nature, 294, p536 (1981); G. F. Hollis et al., Nature, 296, p321 (1982);B. Blomberg et al., Proc. Natl. Acad. Sci. USA, 79, p530 (1982); J.Miller et al., Nature, 295, p428 (1982)!, there is expected an existenceof several subclasses in feline λ chain. In fact, Southern hybridizationusing the λ chain gene of the present invention and a feline chromosomalDNA shows an existence of C region genes of several subclasses belongingto the same feline λ chain gene in addition to the λ chain of thepresent invention. That is, there exist genes of different subclasseshaving a quite similar sequence to that of the gene fragment coding forthe constant region of the feline immunoglobulin λ chain of the presentinvention. The feline λ chain gene of the present invention is believedto have sequences which cover also such gene fragments coding for aminoacid sequences of different subclasses. Therefore, such gene fragmentscoding for different subclasses can also be used as the feline λ chaingene as far as they have substantially the same sequence as that of thegene of the present invention. Cloning of such C genes of differentsubclasses can be carried out by using a part of the nucleotide sequencedisclosed in the present invention as a probe. Alternatively, achromosomal gene coding for the feline λ chain can also be cloned fromfeline cells using the feline immunoglobulin λ chain of the presentinvention as a probe.

κ Chain of immunoglobulin has also already been found in human and mouseP. A. Hieter et al., Cell, 22, p197 (1980); H. Sakano et al., Nature280, p288-294 (1979)! and also reported for other animal species such asrabbit L. Emorine et al., Proc. Natl. Acad. Sci. USA, 80, p5709-5713(1983)!. However, there has never been reported for the feline κ chainof the present invention, an amino acid sequence thereof and anucleotide sequence coding therefor.

The present inventors have obtained a chromosomal DNA fragment whichwill code for the constant region of feline immunoglobulin κ chain bythe above-mentioned procedure. The present inventors have also analyzedthe nucleotide sequence of the chromosomal DNA fragment and havedetermined an amino acid sequence of the constant region. As a result ofcomparison of the amino acid sequence with those of the constant regionof immunoglobulin κ chain derived from human, mouse, rabbit, etc., ithas been found that the gene fragment of the present invention is a genefragment coding for the constant region of immunoglobulin κ chain.

The gene fragment coding for the constant region of felineimmunoglobulin κ chain of the present invention is a DNA sequence codingfor a peptide comprising 109 amino acids and characterized in that fouramino acids at the C-terminus thereof is -Cys-Gln-Arg-Glu. It is knownthat the C-terminus of the amino acid sequence of human or mouse κ chainconstant region is Cys (cysteine) residue as previously reported. It isvery rare that the amino acid sequence of the constant region containsadditional three amino acids subsequent to the Cys residue present atthe C-terminus like in the gene of the present invention coding for theconstant region of feline immunoglobulin κ chain, which ischaracteristic for the constant region of feline immunoglobulin κ chainof the present invention. The gene coding for the constant region offeline immunoglobulin κ chain of the present invention contains the genefragment as shown by the restriction enzyme map of FIG. 10.

Among the genes coding for the constant region of feline immunoglobulinκ chain of the present invention, one of the preferable examples is thegene fragment coding for the amino acid sequence as shown in FIG. 12.Such amino acid sequence or nucleotide sequence coding therefor hashitherto never been reported.

One example of the nucleotide sequence of the gene coding for the Cregion of feline immunoglobulin κ chain of the present invention is asshown in FIG. 11. cDNA coding for the C region of immunoglobulin κ chaincan be cloned from a cDNA library of feline antibody-producing cellsusing the feline immunoglobulin κ chain of the present invention as aprobe.

γ Chain of immunoglobulin has also already been found in human and mousee.g. A. Shimizu et al., Cell, 29, p121 (1982); N. Takahashi et al.,Cell, 29, p671 (1982)! and also reported for other animal species suchas rabbit C. L. Martens et al., Proc. Natl. Acad. Sci. USA, 79, p6018(1982)! and bovine K. L. Knight et al., J. Immunol, 140, p3654 (1988)!.However, there has never been any report concerning the felineimmunoglobulin γ chain.

The present inventors have obtained a chromosomal gene fragment whichwill code for the constant region of feline immunoglobulin γ chain bythe aforementioned procedure. The present inventors have also clonedcDNA gene coding for the C region of immunoglobulin γ chain from a cDNAlibrary of feline antibody-producing cells using the above chromosomalgene fragment. The cloned gene fragment has genetically been analyzed bycomparing an amino acid sequence deduced from the nucleotide sequence ofthis gene fragment with those of the genes derived from other animalspecies coding for the C region of immunoglobulin, and as a result, ithas been found that the gene fragment obtained by the present inventionis a gene fragment coding for the constant region of immunoglobulin γchain.

The obtained DNA fragment coding for the constant region of felineimmunoglobulin γ chain has been analyzed for its nucleotide sequence andan amino acid of the constant region has been determined therefrom. Theamino acid sequence has been compared with previously reported aminoacid sequences of the constant region of immuno-globulin γ chain derivedfrom human, mouse, rabbit, etc. As a result, it has been found that theamino acid sequence specific for the constant region of felineimmunoglobulin γ chain includes the following amino acid sequence (B) inthe vicinity of the first cysteine residue counted from the N-terminusof CH1 domain of the constant region of the γ chain:

    -Ser-Cys-Gly-Thr-                                          (B)

The present inventors have found that an amino acid sequence of theabove region -Ser-Cys-Gly-Thr- present in the vicinity of theabove-mentioned cysteine varies with animal species such as cat, mouse,human, etc. by comparing the amino acid sequence of the constant regionof feline immunoglobulin γ chain analyzed by the present invention withthe previously analyzed amino acid sequences of the constant region ofimmunoglobulin γ chain derived from various animals. The presentinventors have also found that this region is quite well preserved amongsubclasses, for example, as an amino acid sequence of the constantregion of human γ chain. The above sequence (B) is supposed to bespecific for the constant region of feline immunoglobulin γ chain.

Similar sequences specific for the constant region of felineimmunoglobulin γ chain have also been found in the vicinity of cysteineat the C-terminal region of the CH1 domain the following sequence (C)!;at the N-terminal region of the glycosilated site (Asn) of the CH2domain the following sequence (D)!; and in the vicinity of cystein atthe N-terminal region of the CH3 domain the following sequence (E)!:

    -Arg-Trp-Leu-Ser-Asp-Thr-Phe-Thr-Cys-                      (C)

    -Lys-Thr-Ser-Pro-XXX-XXX-XXX-XXX-XXX-Asn                   (D)

    -Asn-Lys-XXX-XXX-XXX-XXX-Cys-                              (E)

wherein XXX represents an optional amino acid.

The amino acid sequences of these regions cloned by the presentinvention have the following sequences (C'), (D') and (E') which areeach one of the preferable examples of specific amino acid sequencespresent in the constant region of feline immunoglobulin γ chain.

    -Arg-Trp-Leu-Ser-Asp-Thr-Phe-Thr-Cys-                      (C')=(C)

    -Lys-Thr-Ser-Pro-Arg-Glu-Glu-Gln-Phe-Asn                   (D')

    -Asn-Lys-Val-Ser-Val-Thr-Cys-                              (E')

The gene fragment coding for the constant region of the felineimmunoglobulin γ chain of the present invention is characterized by thatit contains a DNA sequence coding for the above amino acid sequence (B),(C), (D) or (E). These amino acid sequences contained in the γ chain,which are quite important for determining the C region of the felineimmunoglobulin γ chain, have been found by the present inventors for thefirst time.

A preferable example of the constant region of the feline immunoglobulinγ chain containing these amino acid sequences has an amino acid sequenceas shown in FIG. 18. Such amino acid sequence and a nucleotide sequencecoding therefor have never been reported until the present invention.

An example of the nucleotide sequence of the gene coding for the Cregion of the feline immunoglobulin γ chain of the present inventionincludes the sequence as shown in FIG. 17.

On the analogy of the human and mouse cases for example, A. Shimizu etal., Cell, 29, p121 (1982); N. Takahashi et al., Cell, 29, p671 (1982)!,the feline γ chain is also expected to have several subclasses. It isknown that there are at least two subclasses of feline γ chain J. M.Kehoe et al., J. Immunol., 109, p511 (1972)!, the gene of the presentinvention appears to code for one of these two subclasses. In fact,Southern hybridization using the γ gene of the present invention and afeline chromosomal DNA showed an existence of several genes coding forthe C regions of other subclasses of the feline γ chain in addition tothe γ chain of the present invention. That is, there are genes codingfor different subclasses having extremely similar sequences to that ofthe gene of the present invention. It is believed that the feline γchain gene of the present invention has sequences which cover also suchgene fragments coding for amino acid sequences of different subclasses.Therefore, such gene fragments coding for amino acid sequences ofdifferent subclasses can also be used as feline γ chain gene as far asthey have substantially the same sequence as that of the gene of thepresent invention. Such genes coding for C regions of differentsubclasses can be cloned by using a part of the nucleotide sequence ofthe present invention as a probe.

As far as the constant region of immunoglobulin is concerned, it is alsoreported that a genetic analysis of immunoglobulins derived from human,rabbit, etc. showed an existence of an allotype which differs in one toseveral amino acids within the same class or subclass. Based on theanalogy of this fact, it is expected that there exists an allotype forthe gene fragment coding for the constant region of the immunoglobulin γchain of the present invention. Therefore, the gene coding for theconstant region of the feline immunoglobuin λ chain, κ chain or γ chainof the present invention is not limited to the gene fragments coding forthe above-mentioned amino acid sequences but includes those genes codingfor the constant region of different allotypes which have almost thesame sequence as the above sequences though containing a partialsubstitution of an amino acid.

The thus prepared gene coding for the constant region of felineimmunoglobulin of the present invention allows for preparation of thefeline chimeric antibody using the conventional process for preparingthe chimeric antibody for example, Watanabe et al., Cancer Research, 47,p999-1005 (1987); Japanese Patent First Publication No. 20255/1988!.That is, the chimeric gene can basically be constructed by linking twokinds of gene fragments, i.e. the V region gene and the C region gene,to each other. Since the gene can be classified into mainly twocategories depending on a way of isolation, the chimeric antibody can beconstructed by using either a combination of V and C region genesisolated from a chromosomal DNA or a combination of V and C region genesisolated from cDNA. The chimeric antibody can be expressed in anyexpression system such as an animal cell expression system, an E. coliexpression system, an yeast expression system, etc. using differentexpression vectors.

In a combination of the V region and the constant region for preparingthe chimeric antibody, a preferable combination is the VH region withthe constant region derived from feline H chain such as γ chain, the Vκregion with the constant region of feline κ chain, and the Vλ regionwith the constant region of feline λ chain, but another combination canalso be used. There can be used any V region including those derivedfrom mouse, cat, or other animals, and CDR graft V region M. Verhoeyen,C. Milstein, G. Winter, Science, 239, p1539 (1987)!, as far as it iseffective for treating feline diseases (e.g. viral feline diseases,etc.). Thus, although it is described as to the mouse-cat chimericantibody against feline parvovirus to exemplify the feline chimericantibody of the present invention, the V region is not limited to thatused therein.

The gene fragment coding for feline immunoglobulin provided by thepresent invention discloses the specific amino acid sequence or DNAsequence in the constant region of feline immunoglobulin, and hence,allows for isolating those genes coding for the constant regionsbelonging to different subclasses or allotypes. By using the gene codingfor the constant region of feline immunoglobulin of the presentinvention, the feline chimeric antibody can firstly be prepared. Thefeline chimeric antibody prepared according to the present invention canbe used as agents for diagnosis, prevention and treatment of felinediseases which have hitherto never been developed.

The present invention is illustrated by the following Examples in moredetail but should not be construed to be limited thereto.

EXAMPLE 1

Preparation of Feline Monoclonal Antibody-Producing Cells

(1) Immunization and Preparation of Feline Lymphocytes

In order to efficiently obtain activated feline B cells, a completeFreund's adjuvant (CFA: manufactured by Difco)(5 ml) was injected incats subcutaneously and intraperitoneally for several times at aninterval of 2 to 3 weeks for non-specific immunization. Two to threeweeks after the final immunization, spleen and lymph node were taken outand a suspension of feline lymphocytes was obtained by crashing with apincette and pipetting. One cat gave 1 to 5×10⁹ cells from spleen and 1to 5×10⁸ cells from lymph node. These lymphocytes were suspended in acomplete medium of RPMI 1640 plus 10% fetal calf serum (manufactured byFlow Laboratories) supplemented with L-glutamine (manufactured by FlowLaboratories) at a concentration of 5 to 10×10⁵ cells/ml and thereto wasadded poke-weed mitogen (PWM: manufactured by Gibco)(2.5 μg/ml),followed by culturing the cells in the presence of 5% CO₂ at 37° C. for2 to 5 days for activation.

(2) Preparation of Mouse Myeloma Cells

The myeloma cells employed in the present invention are those derivedfrom mouse Balb/c as described in Koller et al., Nature, 256, p459(1975) and Eur. J. Immunol., 6, p292 (1976), especially substrainsX63-Ag8-6.5.3 and P3-X-63-Ag8-U1, SP2/OAg12. These cells were culturedand grown in a complete medium of RPMI 1640 plus 10% fetal calf serumsupplemented with glutamine. They were collected just before cellfusion, washed twice with RPMI 1640 medium and resuspended in the samemedium for use in cell fusion.

(3) Cell Fusion of the Feline Lymphocytes and the Mouse Myeloma Cells

The above feline lymphocytes and the mouse myeloma cells were mixed at aratio of 10:1 or 5:1 (feline lymphocytes: myeloma cells; felinelymphocytes=1×10⁸ cells) and centrifuged at 1,500 rpm for 5 minutes. Tothe obtained cell pellet was added a 45% polyethylene glycol solutiondiluted with RPMI 1640 (manufactured by Sigma, pH 7.6, MW 3650; ormanufactured by Celba, pH 7.6, MW 4,000)(1 ml) at room temperature over1 minute. After the mixture was allowed to stand at 37° C. for 5 to 10minutes, the cells were resuspended by adding RPMI 1640 (40 ml) to themixture over 6 minutes to quench the cell fusion. The cells were thencentrifuged at 1,000 rpm for 10 minutes, the supernatant was removed bysuction and the resulting cell pellet was resuspended in RPMI 1640+10%fetal calf serum+HAT (H: hypoxanthine 13.0 μg/ml, A: aminopterin 0.18μg/ml, T: thymidine 3.87 μg/ml; all manufactured by Sigma) supplementedwith glutamine at a concentration of 2 to 10×10⁵ lymphocytes/ml. Thesuspension was poured into each well of a 96-well microtiter plate at200 μl/well and was cultured in the presence of 5% CO₂ at 37° C. After 5to 7 days, 50% of the medium was exchanged with the same fresh mediumand thereafter the medium exchange was repeated for 5 to 6 times after10 to 28 days from the cell fusion. By this procedure, only hybridomaswere grown, and thereafter the culture was continued until screeningassay.

(4) Screening Aassay and Cloning of Hybridomas

Screening assay was conducted to detect a clone which produced a felineIgG antibody using enzyme immunoassay (EIA). A 96-well plate was coatedwith a goat anti-feline IgG antibody (manufactured by Cappel) andtreated with bovine serum albumin (manufactured by Sigma) to blocknon-specific adsorption and to each well was added the culturesupernatant (50 μl) from each well of the hybridoma culture plate. Afterincubating the plate at 37° C. for 1 to 2 hours, washing was carried outwith PBS-T 0.01% Tween manufactured by Katayama Kagaku K. K., 0.01Mphosphate buffer, pH 7.2, 0.15M NaCl! four times, a peroxidase-labelledgoat anti-feline IgG antibody (manufactured by Cappel, 10,000 folddilution)(50 μ) was added to each well and the plate was incubated at37° C. for 1 hour. After washing with PBS-T five times, a TMBZ substratesolution (TMBZ: manufactured by Dojin Kagaku K. K., 0.4 mg/ml, hydrogenperoxide: manufactured by Mitsubishi Gasu Kagaku K. K., 0.006%)(50 μ) todevelop color. After 10 to 15 minutes, 0.3N H₂ SO₄ (manufactured byKatayama Kagaku K. K.)(50 μl) was added to each well to quench thereaction and each color development was quantitated with aspectrophotometer (wavelength: 450 nm). The hybridomas in the thusselected feline IgG-producing well were then monoclonalized (cloned) bya limiting dilution procedure. After growth of the clones in each wellof the 96-well plate, the above enzyme immunoassay (EIA) was repeated todetect the feline IgG-producing clone. This cloning procedure wasrepeated at least 3 times to give the cat-mouse hybridoma clone whichstably produces feline IgG. This hybridoma clone was successivelysubjected to an extensive culture and was kept in the freezed state witha cell-freezing medium of RPMI 1640+HAT+10% DMSO (manufactured by WakoPure Chemical Industries) supplemented with glutamine in liquidnitrogen.

(5) Characterization of the Established Hybridoma Clone and Feline IgGMonoclonal Antibody Produced Therefrom

Measurement with an EIA procedure of an amount of the produced felineantibody confirmed that the established cat-mouse hybridomas werecapable of stably producing the feline IgG monoclonal antibody in anamount of 1.0 to 3.0 μg/ml for a long time of 1 year or more and anyabnormality was not observed in their ability of producing the antibody.

The feline monoclonal antibody produced by the established cat-mousehybridoma was proved to be feline IgG by an immunoprecipitation methodcf. "Men-eki Jikken Sosaho (Immunological Experimentation)" ed. Jap.Soc. Immunol.!. This antibody was found to be feline IgG antibody sinceit did not form precipitates with goat anti-mouse IgG serum and goatanti-human IgG serum but did form precipitates with goat anti-feline IgGserum. EIA further proved that the monoclonal antibody was feline IgGantibody since it specifically reacted with only the anti-feline IgGantibody but reacted with neither the anti-human IgG antibody nor theanti-mouse IgG antibody (FIG. 1). Furthermore, Western blotting assaycf. "Men-eki Jikken Sosaho (Immunological Experimentation)" ed. J. Soc.Immunol.! proved that both the heavy chain (H chain) fragment and lightchain (L chain) fragment of the monoclonal antibody specifically reactedonly with the anti-feline IgG antibody but did not react with theanti-human IgG antibody or the anti-mouse IgG antibody and that the Hchain fragment and the L chain fragment of the monoclonal antibody wereidentical to those fragments of standard feline IgG antibody in view ofmolecular weight, showing that the monoclonal antibody is a completefeline IgG monoclonal antibody having both H chain and L chain fragmentsof the feline IgG antibody (FIG. 2).

In addition, cytoplasmic synthesis of feline IgG antibody was studied bycytoplasmic fluorescent antibody dying assay cf. "Men-eki Jikken Sosaho(Immunological Experimentation)" ed. Jap. Soc. Immunol.! of thehybridoma clones. As a result, each clone was intracytoplasmically dyedspecifically only with the anti-feline IgG antibody but was not dyedwith the anti-human IgG antibody and the anti-mouse IgG antibody,proving that the hybridoma clones intracytoplasmically synthesized acomplete feline IgG monoclonal antibody.

The cat-mouse heterohybridoma FM-T1 cells producing such felinemonoclonal antibody has been deposited under accession number BP-2974 asmentioned hereinbefore. The FM-T1 cells were used in the followingExperiment to isolate the constant region of feline immunoglobulin.

EXAMPLE 2

Cloning of the Gene Coding for the Constant Region of FelineImmunoglobulin λ Chain

(1) Construction of a cDNA Library

Total RNA was separated from the heterohybridoma FM-T1 cells using aguanidium thiocyanate method J. M. Ghingwin et al., Biochemistry, 18,p5294 (1979)! and further purified into poly A+RNA using an oligo dTcolumn (Pharmacia). cDNA of the FM-T1 cells was synthesized from thepurified poly A+RNA using a cDNA synthesis system Plus (Amersham). EcoRIsites of the synthesized cDNA were methylated with EcoRI methylase(manufactured by Takara Shuzo Co. Ltd.,; the reagents used hereinafterare manufactured by Takara Shuzo Co. Ltd., or Toyobo Co. Ltd., unlessotherwise mentioned) and then EcoRI linker was added to the cDNA withT4DNA ligase. This cDNA was completely digested with a restrictionenzyme EcoRI and a cDNA having an addition of EcoRI linker was purifiedwith a Bio Gel A50 m column (Bio-Rad). The obtained cDNA was ligated toan EcoRI arm of λgt11 vector DNA (Stratagene) with T4DNA ligase and thenan in vitro packaging was carried out using kits manufactured byStratagene to give a cDNA library of the FM-T1 cells.

(2) Screening of the Feline Immunoglobulin Gene with Anti-Feline IgGAntibody

Using the cDNA library of FM-T1 cells constructed as mentioned above, E.coli Y1090 strain cells infected with the phage λgt11 were poured ontoan LB plate laboratory dish of No.2 square manufactured by Eiken Kizaicharged with 1.5% Bacto-agar manufactured by Gifco, 1% Bacto-tryptonemanufactured by Gifco, 0.5% Bacto-yeast extract manufactured by Gifco,0.25% NaCl manufactured by Wako Pure Chemical Industries, pH 7.5! sothat 50,000 plaques per plate of the λgt11 phage were formed andcultured at 42° C. for 3 hours. After covering a nitrocellulose filter(NC filter: Code BA85 manufactured by S&S) soaked with 10 mM IPTG(manufactured by Wako Pure Chemical Industries) on the plate, theculture was continued at 37° C. for additional 4 hours. The NC filterwas peeled off the plate, washed with a W buffer WB: 7 mM Tris buffer,pH 7.2, 150 mM NaCl, 0.005% Tween 20! and immersed in a BLOTTO 5% skimmilk, 10 μl/100 ml Antifoam A! at 4° C. overnight. Then the BLOTTO wasexchanged with a BLOTTO containing an anti-feline IgG antibodymanufactured by Cappel; 10 μg/ml; treated with a 1% E. coli lysate(manufactured by Bio-Rad) at 4° C. overnight! and the reaction wasconducted at room temperature for 2 hours. After washing the NC filterwith WB five times, the NC filter was immersed in an incubation bufferPBS, pH 7.2, 0.005% Tween 20, 1% BSA! containing a peroxidase-labelledgoat IgG antibody manufactured by Cappel; treated with a 1% E. colilysate (manufactured by Bio-Rad) at 4° C. overnight! and the reactionwas conducted at room temperature for 2 hours. After washing the NCfilter with WB five times, the NC filter was immersed in a colordevelopment solution containing 5% HRP color develoment reagent(manufactured by Bio-Rad) and 0.5% H₂ O₂ (manufactured by Wako PureChemical Industries) to proceed color development. After selection ofthe phage corresponding to the plaque which showed the color developmenton the NC filter, the phage was further cloned. The clone was selectedin this way which reacted with the anti-feline IgG antibody and finallya clone T1-62 was obtained. This clone had a 0.7 kb size and seemed tobe a gene coding for the immunoglobulin λ chain in accordance with thenucleotide sequence analysis as described hereinafter. FIG. 3 shows arestriction enzyme map of this clone. The EcoRI insertion fragment ofthis clone was isolated from the phage DNA in accordance with a methodby Thomas and Davis M. Thomas and R. W. Davis, J. Mol. Biol., 91, p315(1974)! and subcloned into the EcoRI site of pUC18 vector.

(3) Southern Blotting and Northern Blotting Analysis Using T1-62

Southern blotting analysis was carried out using T1-62. A chromosomalDNA (10 μg) from feline, canine and mouse hepatocytes was digested withthe restriction enzyme EcoRI and the obtained DNA fragments weresubjected to electrophoresis using 0.7% agarose gel. After transfer to anylon membrane filter (Gene Screen Plus, NEN Research Product), thesouthern hybridization was carried out with a ³² P!-labelled T1-62 probecontaining the constant region of the feline λ chain. The southernhybridization was carried out in accordance with a protocol of a manualattached to the Gene Screen Plus. The molecular size was calculatedbased on a marker DNA which was prepared by digesting the λ phage DNAwith HindIII. As shown in FIG. 4, several bands having various sizesfrom about 2 to about 20 kb were detected. This suggests that the genecoding for the C region of feline λ chain has more than one subclass,which can also be estimated on the analogy of the cases of human andmouse P. A. Hieter et al., Nature, 294 , p536 (1981); G. F. Hollis etal., Nature, 296, p321 (1982); B. Blomberg et al., Proc. Natl. Acad.Sci. USA, 79, p53 (1982); J. Miller et al., Nature, 295, p428 (1982)!.Since it is known that the gene coding for the C λ region is amplifiedin wild mice C. L. Scott et al., Nature, 300, p757 (1982)!, the genecoding for the C region of the feline λ chain can also presumably besubjected to amplification.

Northern blotting was then conducted. The RNA for use in thehybridization was prepared by separating a whole RNA from FM-T1 andFM-T2 cells using a guanidium thiocyanate method J. M. Ghingwin et al.,Biochemistry, 18, p5294 (1979)! and purifying the whole RNA into a polyA+RNA with oligo dT column (Pharmacia). The RNA (2 μg) waselectrophoresed using 0.75% agarose gel containing 3% formaldehyde.After transfer to a nylon membrane filter (Gene Screen Plus), a northernhybridization was carried out with a ³² P!-labelled T1-62 probe. Thenorthern hybridization was carried out in accordance with a protocol ofa manual attached to the Gene Screen Plus. As a result, this probedetected a band at about 1.3 kb in both mRNAs (FIG. 5). This size isalmost the same as that of the gene coding for immunoglobulin λ chainknown in mouse and human.

From the above two results, the T1-62 gene was presumed to be an activegene coding for a functional feline Cλ region.

(4) Nucleotide Sequence and Amino Acid Sequence of T1-62

In order to determine a nucleotide sequence of T1- 62, small DNAfragments EcoRI-SacI, SacI-AccII, AccII-EcoRI and EcoRI-HhaI wereprepared (FIG. 3) from T1-62. These small fragments were blunt-endedwith T4-DNA polymerase and inserted into a SmaI site of a M13mp19 vectorusing a Takara Ligation Kit. Competent cells of JM101 were prepared inaccordance with a Toyobo Instruction Manual and transformed with M13mp19DNA wherein the Cλ gene was inserted, followed by extraction andpurification of a single stranded DNA. A nucleotide sequence of thissingle stranded DNA was determined using a Takara M13 Sequencing Kit andFuji Gencer Gel System. The direction of determination of the nucleotidesequence is shown in FIG. 3. As a result of the determination of thenucleotide sequence, it was confirmed that the feline λ chain geneconsisted of V, J and C regions. FIG. 6 shows the results thereof. Anamino acid sequence deduced from this nucleotide sequence suggested thatthe gene is in an open reading frame and is not a pseudogene (FIG. 7).

A homology of the nucleotide sequence of T1-62 was searched on databases, LASL and EMBL, using a software of genetic analysis (GenetyxVer.6 manufactured by Software Kaihatsu, K. K.). As a result, thehighest homology was shown with that of human immunoglobulin λ chaingene but no homology was shown with that of genes other than theimmuno-globulin λ chain gene. Homological comparison of the Cλ region ofthe T1-62 gene with those of mouse and human showed 75.8% homology withmouse and 81.3% homology with human in the nucleic acid level and 69.5%homology with mouse and 77.1% homology with human in the amino acidlevel.

As is clear from the above results, the T1-62 gene is surely a genecoding for the feline λ chain which can be used for the preparation of afeline chimeric antibody.

EXAMPLE 3

Cloning of the Gene Coding for Constant Region of Feline κ Chain

(1) Conditions of Crosshybridization

The L chain of feline immunoglobulin is known to be mainly consisted ofλ chain L. Hood et al., Cold Spring Harbor Symp. Quant. Biol. 32,p133-146 (1967)!, and hence, it is foreseeable that only quite a fewlymphocytes express κ chain. In fact, the cat-mouse heterohybridomas asdescribed above were λ chain-producing cells. Therefore, the presentinventors have considered that it would be very difficult to obtain thedesired gene from messenger RNAs of polyclonal antibody-producing cellsby a cDNA cloning procedure, and hence, have tried to isolate thedesired gene fragment coding for the constant region of felineimmuno-globulin κ chain from a chromosomal DNA of feline hepatocyteunder crosshybridization conditions using the constant region of human κchain (human Cκ) as a probe.

For cloning of the feline κ chain gene by a cross-hybridizationprocedure, conditions of crosshybridization with human κ chaim werestudied. The gene coding for the human Cκ region used for thecrosshybridization was a gene cloned from a human culture cell ARH77strain ATCC CRL 1621! which is available from professor TakeshiWatanabe, Department of Molecular Immunology, Medical Institute ofBioregulation, Kyushu University cf. Kudo et al., Gene, 33, p181 (1985);Nishimura et al., Cancer Res., 47, p999 (1987)!. From this human Cκgene, an EcoRI--EcoRI fragment containing Cκ exon was obtained and usedas a probe.

Chromosomal DNAs were isolated from feline hepatocytes according to N.Blin and D. W. Stafford Nuc. Acids. Res., 3, p2303 (1976)! and 10 μgeach of the chromosomal DNA was digested with the restriction enzymeEcoRI. DNA fragments obtained by the restriction enzyme digestion weresubjected to electrophoresis using 0.7% agarose gel. The developed DNAswere transferred to a nitrocellulose membrane filter (BA85 manufacturedby S&S) and then southern hybridization was carried out with a ³²P!-labelled DNA probe containing human Cκ region gene. The southernhybridization was carried out in a solution of 6×SSC 0.09M Na₃ C₆ H₅O₇.2H₂ O, 0.9M NaCl!, 10 mM EDTA manufactured by Dojin Kagaku! and 0.5%SDS manufactured by Bio-Rad! at 65° C. overnight. The final washing ofthe filter was conducted with a solution of 0.1×SSC and 0.1% SDS at 45°C. for 15 minutes. Autoradiography of this filter showed a band at about5.5 kb as shown in FIG. 8. The molecular size was calculated from amarker DNA prepared by digesting λ phage DNA with HindIII. This DNAfragment of 5.5 kb appears to contain the gene coding for feline κ chainand was used for cloning.

(2) Isolation of the Gene Coding for Feline κ Chain

A chromosomal DNA (100 μg) from feline hepatocytes was completelydigested with EcoRI and DNA fragments of 5.5 kb were prepared by asucrose density gradient centrifugation sucrose 10 to 40% (wt/vol),26,000 rpm, 18 hours, 15° C.!. The obtained DNA fragments were thenligated to an EcoRI arm of λgt11 vector DNA (manufactured by Stratagene)with T4 DNA ligase and an in vitro packaging was carried out using a kitavailable from Stratagene to give a κ chain gene library of felinehepatocytes. Plaque hybridization W. D. Benton, R. W. Davis, Science,196, p180 (1977)! was conducted using a human Cκ probe under the sameconditions as those of the above crosshybridization and a clone CEκ8acontaining a feline Cκ chain exon was selected from the library. FIG. 10shows a restriction enzyme map of this clone. The EcoRI insertionfragment of this clone was isolated from the phage DNA in accordancewith a method by Thomas and Davis M. Thomas and R. W. Davis, J. Mol.Biol., 91, p315 (1974)! and subcloned into the EcoRI site of pUC18vector.

(3) Southern Blotting and Northern Blotting Analysis using CEκ8a

A chromosomal DNA (10 μg) from feline hepatocytes was digested with therestriction enzyme EcoRI and the Southern blotting analysis wasconducted using the clone CEκ8a in the same manner as described inExample 2 (3). A pattern of the detected bands was compared with thatfrom crosshybridization using a human Cκ chain probe previouslyconducted, and as a result, a band was observed at the same position(about 5.5 kb). This result presumably showed that the feline Cκ regiongene consists of a single gene without any other subtypes. This is alsosuggested by the cases of human and mouse P. A. Hieter et al., Cell, 22,p197 (1989); E. E. Max et al., Proc. Natl. Acad. Sci. USA, 76, p3450(1979)!.

Northern blotting analysis was then carried out using the CEκ8a clone inthe same manner as described in Example 2 (3). As a result, the probedetected a band at about 1.3 kb with mRNA from feline spleen cells (FIG.9). This is almost the same size as known in the mouse and humanimmunoglobulin κ chain gene.

These two results presumably showed that the CEκ8a is an active genecontaining a gene coding for a functional feline Cκ region.

(4) Nucleic Acid and Amino Acid Sequences of the CEκ8a

In order to determine a nucleic acid sequence of the gene coding for thefeline Cκ region, a DNA fragment (XbaI-AvaI fragment) of about 2.0 kbcontaining the Cκ region was isolated from the clone CEκ8a and reclonedinto a XbaI-AvaI site of pUC18 vector. This plasmid was prepared in alarge amount in accordance with the conventional method for example, T.Maniatis "Molecular Cloning" Cold Spring Harbor Lab. (1982)! and fromthe XbaI-AvaI fragments were prepared small DNA fragments (XbaI-DdeI,DdeI-HaeIII, HaeIII-HinfI, XbaI-RsaI). These small DNA fragments wereblunt-ended with T4-DNA polymerase and then inserted into a SmaI site ofM13mp19 vector using a Takara Ligation Kit. The nucleotide sequence wasdetermined in the same manner as described in Example 2 (4). A directionof determination of the nucleotide sequence is shown in FIG. 10. As aresult of the determination of the nucleic acid base sequence, the Cκgene comprising a single exon was confirmed. FIG. 11 shows the resultsthereof. An amino acid sequence deduced from the nucleotide sequenceshowed that the gene is in an open reading frame and is not a pseudogene(FIG. 12).

A homology of the nucleotide sequence of CEκ8a2 was searched on databases, LASL and EMBL, using a software of genetic analysis (Genetyxmanufactured by Software Kaihatsu, K. K.). As a result, a high homologywas shown with genes coding for human and mouse immunoglobulins κ chainbut no homology was shown with genes other than the immunoglobulin κchain gene. Homological comparison of the Cκ region of the CEκ8a genewith the mouse and human Cκ regions showed 73.3% homology with mouse and73.0% homology with human in the nucleic acid level and 54.7% homologywith mouse and 59.6% homology with human in the amino acid level.

As is clear from the above results, the CEκ8a gene is surely a genecoding for the feline κ chain which can be used for the preparation ofthe cat-mouse chimeric antibody.

EXAMPLE 4

Cloning of the Gene Coding for Constant Region of Feline γ Chain

(1) Conditions of Crosshybridization

The present inventors then tried to isolate the feline γ chain gene bycloning using a cross-hybridization procedure as in the case of κ chaingene.

In the same manner as in Example 3 (1), cross-hybridization was carriedout using a chromosomal DNA (10 μg) from feline hepatocytes digestedwith the restriction enzyme BamHI and a human Cγ1 chain as a probe. Thegene coding for the human Cγ1 region used for the cross-hybridizationwas a gene cloned from a human culture cell ARH77 strain ATCC CRL 1621!which is available from professor Takeshi Watanabe, Department ofMolecular Immunology, Medical Institute of Bioregulation, KyushuUniversity Kudo et al., Gene, 33, p181 (1985); Nishimura et al., CancerRes., 47, p999 (1987)!. Computerized analysis of homology between mouse,human and rabbit γ chains showed an extremely high homology especiallyat the region containing a CH2-CH3 exon. Therefore, a PstI-SphI fragmentcontaining the CH2-CH3 exon was obtained from the human Cγ1 gene andused as a probe. The result of autoradiography showed two bands at about5.8 kb and 5.5 kb as shown in FIG. 13. These bands were targetted forcloning.

(2) Isolation of the Feline γ Chain Gene

A chromosomal DNA (100 μg) from feline hepatocytes was completelydigested with BamHI and then DNA fragments corresponding to the above5.5 kb and 5.8 kb were prepared by a sucrose density gradientcentrifugation sucrose 10 to 40% (wt/vol), 26,000 rpm, 18 hours, 15°C.!. Then, the obtained DNA fragments were ligated to a BamHI-digestedDNA of Charomid 9-42 vector DNA (manufactured by Nippon Gene) with T4DNA ligase and an in vitro packaging was carried out using a kitavailable from Stratagene. LE392 E. coli (Stratagene) was then infectedwith the resultant vector DNA and a γ chain gene library of felinehepatocytes was obtained. Colony hybridization up-to-date: IdenshisosaJikken Jitsuyo Handbook (Handbook for Practice of Gene Manipulation)p426! was conducted under the same conditions as those of the abovedescribed crosshybridization and a clone CB25γ7c containing the felineCγ chain exon was selected from the library using the human Cγ1 probe.This clone had a size of about 5.8 kb and was one of two bands shown inthe above Southern blotting analysis. FIG. 14 shows a restriction enzymemap thereof. This plasmid clone was prepared in a large amount inaccordance with the conventional method for example, T. Maniatis"Molecular Cloning" Cold Spring Harbor Lab. (1982)! and a BamHIinsertion fragment was isolated. For easier handling, the fragment wascleaved with SacI and a DNA fragment (SacI fragment) of about 1.3 kbassumed to contain the Cγ exon were isolated and recloned into a SacIsite of pUC18 vector.

(3) Southern and Northern Blotting Analysis Using CB25γ7c

A chromosomal DNA (10 μg) from feline hepatocytes was digested with therestriction enzyme BamHI and Southern blotting analysis was conductedusing the CB25γ7c clone in the same manner as described in Example 2(3). Comparison of a pattern of detected bands with that ofcross-hybridization previously conducted using the human Cγ1 chain probeshowed a band at the same position (about 5.8 kb and 5.5 kb)(the sameresults as in FIG. 13). This result showed an existence of several Cγregion genes of different subclasses belonging to the same feline γchain in addition to the CB25γ7c.

The existence of several subclasses in the feline γ chain is alsosuggested on the analogy of the cases of human and mouse for example, A.Shimizu et al., Cell, 29, p121 (1982); N. Takahashi et al., Cell, 29,p671 (1982)!. It is known that serologically at least two subclassesexist for the feline γ chain J. M. Kehoe, J. Immunol., 109, p511 (1972)!and the CB25γ7c gene seems to code for either of the two subclasses.

Then, Northern blotting analysis was carried out using the CB25γ7c clonein the same manner as described in Example 2 (3). As a result, the probedetected a band at about 1.8 kb with mRNAs from cat-mouseheterohybridoma FM-T1 cells (FIG. 15). This has almost the same size asknown in mouse and human immunoglobulin γ chain genes.

These two results presumably showed that the CB25γ7c clone is an activegene containing a gene coding for a functional feline Cγ region.

(4) Isolation of Feline γ Chain cDNA Clone

It is presumed that the feline Cγ region has an exon-intron structure onthe analogy of the mouse and human cases. In order to determine anucleotide sequence of the exon part of the feline Cγ region, it isnecessary to clone a cDNA from mRNA of feline immunoglobulin γ chainwhere splicing has been completed. As mentioned above, the cat-mouseheterohybridoma FM-T1 cells synthesized a mRNA hybridizable with theCB25γ7c clone. Therefore, the present inventors tried to clone a cDNA offeline γ chain from a cDNA library of FM-T1 cells using the CB25γ7c as aprobe. The construction of the cDNA library of FM-T1 cells has alreadybeen described in Example 2 (1). From this library, a clone T1CB1acontaining cDNA coding for feline immuno-globulin Cγ chain is selectedby a plaque hybridization procedure W. D. Benton, R. W. Davis, Science,196, p180 (1977)! using the CB25γ7c as a probe. This clone has a 1.5 kbsize and a restriction enzyme map thereof is shown in FIG. 16. An EcoRIinsertion fragment of this clone was isolated from the phage DNA inaccordance with Thomas and Davis W. Thomas, R. W. Davis, J. Mol. Biol.,91, p315 (1974)! and recloned at the EcoRI site of pUC18.

(5) Nucleotide Sequence and Amino Acid Sequence of T1CB1a

In order to determine a nucleotide sequence of the gene coding forfeline Cγ region, small DNA fragments (PstI--PstI, PstI-RsaI,PstI-HindIII, SacI-SmaI, EcoRI-SacI, HindIII-EcoRI) were prepared. Thesesmall fragments were blunt-ended using T4 DNA polymerase and theninserted at the SmaI site of M13mp19 vector using a Takara Ligation Kit.The nucleotide sequence was determined in the same manner as describedin Example 2 (4). A direction to determine the nucleotide sequence isshown in FIG. 16. As a result of the determination of the nucleotidesequnece, it was confirmed that the feline γ gene comprised V, D, J andC. FIG. 17 shows the results thereof. An amino acid sequence was thendeduced from the nucleotide sequence, and as a result, it was shown thatthe gene was in an open reading frame and was not a pseudogene (FIG.18).

Homology of the nucleotide sequence of T1CB1a was searched on databases, LASL and EMBL, using a software for genetic analysis (Genetyxmanufactured by Software Kaihatsu K. K.). As a result, a high homologywas shown with genes coding for human and mouse immunoglobulin γ chainsbut no homology was shown with genes other than immunoglobulin γ chaingene. Homological comparison of the Cγ region of T1CB gene with themouse and human Cγ region genes showed 70.2% homology with mouse γ1 and76.8% homology with human G1 in the nucleotide sequence level and 61.0%homology with mouse γ1 and 69.7% homology with human G1 in the aminoacid sequence level.

The above results confirmed that the CB25γ7c and T1CB1a genes weresurely genes coding for feline γ chain which can be used for a mouse-catchimeric antibody.

EXAMPLE 5 Preparation of Mouse-Cat Chimeric Antibody

(1) Isolation of Gene Coding for Mouse Immuno-Globulin κ Chain Variable(Vκ) Region

In order to show usefulness of the thus isolated gene coding for theconstant region of feline immunoglobulin in the preparation of thechimeric antibody, a chimeric antibody comprising said gene and a genecoding for the variable region of mouse antibody JP2 having aneutralization activity against canine parvovirus (CPV) was prepared. Itis known that a monoclonal antibody capable of neutralizing canineparvovirus can also neutralize feline parvovirus because of the highhomology between the canine and feline parvoviruses.

The gene coding for the Vκ region of JP2 antibody was firstly isolated.A chromosomal DNA (100 μg) isolated from hybridomas JP2(γ1,κ) producingan anti-CPV antibody was digested with the restriction enzyme HindIII.This DNA fragment was then ligated to λL47 vector DNA (Amersham) with T4DNA ligase and a chromosomal DNA library of JP2 cells was obtained. Aclone JP2gL411 containing the Vκ region gene of the anti-CPV antibodywas selected from the library by a plaque hybridization procedure W. D.Benton, R. W. Davis, Science, 196, p180 (1977)! using a mouse Jκ probeE. E. Max et al., J. Biol. Chem., 256, p5116 (1981)!. FIG. 19 shows arestriction enzyme map thereof. From this gene fragment there wasprepared a BamHI-HindIII fragment containing a Vκ exon part and thefragment was subjected to Northern hybridization with mRNAs of JP2 andits mother strain P3X63Ag8.U1 to detect a JP2 specific band at 1.3 Kbp.A nucleotide sequence was determined (FIG. 20) by a DNA sequencing whichwas made at a direction of an arrow as shown in FIG. 19. An amino acidsequence deduced from the nucleotide sequence was in an open readingframe, and hence, the gene was shown to code for a functionalimmunoglobulin Vκ. Based on these results, this Vκ gene was used forpraparing a gene coding for an L chain of a mouse-cat chimeric antibody.

(3) Preparation of Gene Coding for L Chain of Mouse-Cat ChimericAntibody (pSV2-EPLCCκ)

The plasmid pCEκ8aXA prepared in Example 3 (2) was digested with HindIIIand EcoRI to prepare a HindIII-EcoRI DNA fragment of 2 kb containing agene coding for feline immunoglobulin Cκ chain. On the other hand, aplasmid pJP2gL411 containing the gene coding for mouse immuno-globulinVκ chain prepared in Example 5 (1) was digested with BamHI and HindIIIto prepare a gene of 4.4 kb coding for a mouse immunoglobulin Vκ-Jκregion. These genes were ligated to each other together with pSV2-neovector P. J. Southern et al., J. Mol. Appl. Genet., 1, p327 (1982)!which was digested with EcoRI and BamHI using a Takara Ligation Kit toprepare a plasmid pSV2-PLCCκ. The human heavy chain enhancer element wasinserted into the HpaI site of this plasmid to prepare a plasmidpSV2-EPLCCκ (FIG. 21).

(5) Expression of Mouse-Cat Chimeric Antibody

The constructed plasmid pSV2-EPLCCκ was introduced into a mouse Blymphocyte strain Sp2/0-Ag12 (ATCC CRL 1581) in accordance with a methodpreviously reported by Maeda et al. Japanese Patent First PublicationNo. 20255/1988! using a DEAE-dextran method. The cells were tranformedwith the plasmid pSV2-EPLCCκ to prepare cells which produce the desiredL chain of the mouse-cat chimeric antibody.

As mentioned above, only use of the gene coding for the constant regionof feline immunoglobulin as cloned by the present inventors makes itpossible to prepare the feline chimeric antibody. The thus preparedfeline chimeric antibody can be used as agents for diagnosis, preventionand treatment of feline disease, especially feline infectious diseases.

What is claimed is:
 1. A recombinant DNA molecule coding for a mouse-catchimeric antibody H chain which comprises a gene fragment coding for thefollowing amino acid sequence of a constant region of felineimmunoglobulin γ chain and a gene fragment coding for an amino acidsequence of a variable region of mouse immunoglobulin H chain whereinthe former gene fragment is linked to the 3' site of the latter genefragment:-Thr-Thr-Ala-Pro-Ser-Val-Phe-Pro-Leu-Ala-Pro-Ser-Cys-Gly-Thr-Thr-Ser-Gly-Ala-Thr-Val-Ala-Leu-Ala-Cys-Leu-Val-Leu-Gly-Tyr-Phe-Pro-Glu-Pro-Val-Thr-Val-Ser-Trp-Asn-Ser-Gly-Ala-Leu-Thr-Ser-Gly-Val-His-Thr-Phe-Pro-Ala-Val-Leu-Gln-Ala-Ser-Gly-Leu-Tyr-Ser-Leu-Ser-Ser-Met-Val-Thr-Val-Pro-Ser-Ser-Arg-Trp-Leu-Ser-Asp-Thr-Phe-Thr-Cys-Asn-Val-Ala-His-Pro-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Thr-Val-Arg-Lys-Thr-Asp-His-Pro-Pro-Gly-Pro-Lys-Pro-Cys-Asp-Cys-Pro-Lys-Cys-Pro-Pro-Pro-Glu-Met-Leu-Gly-Gly-Pro-Ser-Ile-Phe-Ile-Phe-Pro-Pro-Lys-Pro-Lys-Asp-Thr-Leu-Ser-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Leu-Val-Val-Asp-Leu-Gly-Pro-Asp-Asp-Ser-Asp-Val-Gln-Ile-Thr-Trp-Phe-Val-Asp-Asn-Thr-Gln-Val-Tyr-Thr-Ala-Lys-Thr-Ser-Pro-Arg-Glu-Glu-Gln-Phe-Asn-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Pro-Ile-Leu-His-Gln-Asp-Trp-Leu-Lys-Gly-Lys-Glu-Phe-Lys-Cys-Lys-Val-Asn-Ser-Lys-Ser-Leu-Pro-Ser-Pro-Ile-Glu-Arg-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-His-Glu-Pro-Gln-Val-Tyr-Val-Leu-Pro-Pro-Ala-Gln-Glu-Glu-Leu-Ser-Arg-Asn-Lys-Val-Ser-Val-Thr-Cys-Leu-Ile-Lys-Ser-Phe-His-Pro-Pro-Asp-Ile-Ala-Val-Glu-Trp-Glu-Ile-Thr-Gly-Gln-Pro-Glu-Pro-Glu-Asn-Asn-Tyr-Arg-Thr-Thr-Pro-Pro-Gln-Leu-Asp-Ser-Asp-Gly-Thr-Tyr-Phe-Val-Tyr-Ser-Lys-Leu-Ser-Val-Asp-Arg-Ser-His-Trp-Gln-Arg-Gly-Asn-Thr-Tyr-Thr-Cys-Ser-Val-Ser-His-Glu-Ala-Leu-His-Ser-His-His-Thr-Gln-Lys-Ser-Leu-Thr-Gln-Ser -Pro-Gly-Lys.
 2. Therecombinant DNA molecule according to claim 1 wherein the gene fragmentcoding for the amino acid sequence of a constant region of felineimmunoglobulin γ chain comprises the following nucleotide sequence:ACCACG GCC CCA TCG GTG TTC CCA CTG GCC CCC AGC TGC GGG ACC ACA TCT GGC GCCACC GTG GCC CTG GCC TGC CTG GTG TTA GGC TAC TTC CCT GAG CCG GTG ACC GTGTCC TGG AAC TCC GGC GCC CTG ACC AGC GGT GTG CAC ACC TTC CCG GCC GTC CTGCAG GCC TCG GGG CTG TAC TCT CTC AGC AGC ATG GTG ACA GTG CCC TCC AGC AGGTGG CTC AGT GAC ACC TTC ACC TGC AAC GTG GCC CAC CCG CCC AGC AAC ACC AAGGTG GAC AAG ACC GTG CGC AAA ACA GAC CAC CCA CCG GGA CCC AAA CCC TGC GACTGT CCC AAA TGC CCA CCC CCT GAG ATG CTT GGA GGA CCG TCC ATC TTC ATC TTCCCC CCA AAA CCC AAG GAC ACC CTC TCG ATT TCC CGG ACG CCC GAG GTC ACA TGCTTG GTG GTG GAC TTG GGC CCA GAT GAC TCC GAT GTC CAG ATC ACA TGG TTT GTGGAT AAC ACC CAG GTG TAC ACA GCC AAG ACG AGT CCG CGT GAG GAG CAG TTC AACAGC ACC TAC CGT GTG GTC AGT GTC CTC CCC ATC CTA CAC CAG GAC TGG CTC AAGGGG AAG GAG TTC AAG TGC AAG GTC AAC AGC AAA TCC CTC CCC TCC CCC ATC GAGAGG ACC ATC TCC AAG GCC AAA GGA CAG CCC CAC GAG CCC CAG GTG TAC GTC CTGCCT CCA GCC CAG GAG GAG CTC AGC AGG AAC AAA GTC AGT GTG ACC TGC CTG ATCAAA AGC TTC CAC CCG CCT GAC ATT GCC GTC GAG TGG GAG ATC ACC GGA CAG CCGGAG CCA GAG AAC AAC TAC CGG ACG ACC CCG CCC CAG CTG GAC AGC GAC GGG ACCTAC TTC GTG TAC AGC AAG CTC TCG GTG GAC AGG TCC CAC TGG CAG AGG GGA AACACC TAC ACC TGC TCG GTG TCA CAC GAA GCT CTG CAC AGC CAC CAC ACA CAG AAATCC CTC ACC CAG TCT CCG GGT AAA.